乙醛脱氢酶2调控巨噬细胞M2型极化促进创面愈合

张思敏; 亓发芝

doi:10.12025/j.issn.1008-6358.2024.20240828

乙醛脱氢酶2调控巨噬细胞M2型极化促进创面愈合

Aldehyde dehydrogenase 2 promotes wound healing by regulating M2 macrophage polarization

摘要

摘要:
目的验证乙醛脱氢酶2（aldehyde dehydrogenase 2，ALDH2）在创面愈合中的作用并探讨其分子机制。
方法构建小鼠全层皮肤切除模型，随机分为对照组和Alda-1（ALDH2激动剂）组，观察创面愈合情况并检测创面组织ALDH2活性。采用Masson染色观察创面胶原纤维含量，免疫组化染色检测ALDH2和α-平滑肌肌动蛋白（α-smooth muscle actin, α-SMA）水平，免疫荧光染色检测Ⅰ型胶原（Col-Ⅰ）、Ⅲ型胶原（Col-Ⅲ）表达和F4/80^＋、iNOS^＋、CD206^＋细胞数。体外实验采用白介素4（interleukin-4, IL-4）诱导RAW264.7细胞M2型极化，分别用Alda-1和ALDH2抑制剂CVT-10216处理细胞。流式细胞仪检测F4/80^＋CD206^＋细胞比例，酶联免疫分析测定细胞上清中促炎因子和抗炎因子水平，Western印迹法检测AKT/mTOR信号通路相关蛋白表达。
结果与对照组相比，Alda-1组小鼠创面愈合率显著提高，创面的Col-Ⅰ、Col-Ⅲ及α-SMA表达增多；F4/80^＋细胞差异无统计学意义，iNOS^＋细胞显著减少，CD206^＋细胞显著增多。体外实验结果显示，与IL-4组相比，IL-4+Alda-1组F4/80^＋CD206^＋细胞比例、抗炎因子和p-AKT、p-mTOR蛋白表达显著升高，促炎因子表达降低；IL-4+CVT-10216组F4/80^＋CD206^＋细胞比例、抗炎因子和p-AKT、p-mTOR蛋白表达显著降低，促炎因子表达升高。
结论 ALDH2通过AKT/mTOR通路诱导巨噬细胞M2型极化，促进小鼠创面愈合。

Abstract:
Objective To verify the role of aldehyde dehydrogenase 2 (ALDH2) in wound healing and to explore the underlying mechanisms.
Methods A murine excisional wound model was developed and mice were randomly assigned to control group and Alda-1 (ALDH2 agonist) group. Wound healing rate and activity of ALDH2 were measured. Masson staining was used to observe the collagen fiber content, and immunohistochemistry was used to detect the expression of ALDH2 and α-smooth muscle actin (α-SMA). The expression of collagen type Ⅰ and Ⅲ (Col-Ⅰ, Col-Ⅲ), and the number of F4/80, inducible nitric oxide synthase (iNOS) and CD206 positive (F4/80⁺, iNOS⁺, CD206⁺) cells were analyzed using immunofluorescences. Furthermore, interleukin (IL)-4-stimulated RAW264.7 cells were treated with Alda-1 or CVT-10216 (an ALDH2 inhibitor) in vitro. The proportion of F4/80⁺CD206⁺ cells were analyzed using flow cytometry. Anti-inflammatory and pro-inflammatory cytokines were examined using ELISA. The expression of protein associated with the AKT/mTOR pathway were detected via western blotting.
Results Compared with control group, the wound healing rate of mice in the Alda-1 group was significantly improved, and the expression of Col-Ⅰ, Col-Ⅲ, and α-SMA in the wound surface increased. Although the number of F4/80⁺ cells in wounds did not significantly differ, there was a decrease in iNOS⁺ cells and an increase in CD206⁺ cells. In vitro, compared to IL-4 group, IL-4+Alda-1 group exhibited an increased proportion of F4/80⁺CD206⁺ macrophages and higher levels of anti-inflammatory factors, alongside a reduction in levels of pro-inflammatory factors. Conversely, IL-4+CVT-10216 group demonstrated a decreased proportion of F4/80⁺CD206⁺ macrophages, lower levels of anti-inflammatory factors with a concomitant increase in pro-inflammatory factors. Additionally, the protein expressions of p-AKT and p-mTOR were significantly elevated in IL-4+Alda-1 group and diminished in IL-4+CVT-10216 group.
Conclusion ALDH2 induces M2 macrophage polarization via AKT/mTOR pathway to promote wound healing in mice.

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